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The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy.

ABSTRACT: The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ?24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.

SUBMITTER: Kabbani AM

PROVIDER: S-EPMC5647545 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy.

Kabbani Abir M AM Kelly Christopher V CV

Biophysical journal 20171001 8

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics ...[more]

PMID: 29045872

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Project description:The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.

Dataset Information

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy.

Publications

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy.

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